Composition and method for lactose hydrolysis

ABSTRACT

The present invention relates to a composition for the enzymatic hydrolysis of lactose containing two lactase enzymes having distinct pH optima. The composition is suitable for treating or controlling the symptoms of lactose intolerance in humans.

This application is a divisional of application of Ser. No. 08/543,975,filed Oct. 17, 1995, now U.S. Pat. No. 6,410,018.

FIELD OF THE INVENTION

The present invention relates to a composition and method for theenzymatic hydrolysis of lactose and, more particularly, to treating andcontrolling the symptoms of lactose intolerance.

BACKGROUND OF THE INVENTION

The human digestive system uses a series of enzymes to break downcomplex foods into simple molecules that can be absorbed by the body.Milk products contain lactose, which, when hydrolyzed, yield glucose andgalactose. This hydrolysis is enzymatically catalyzed by lactase orβ-D-galactosidase.

Decreased or non-existent intestinal lactase activity, known as lactoseintolerance, is a deficiency that appears in pediatric, adolescent andadult populations. The inability to hydrolyze lactose into its componentsugars results in bloating, cramping, abdominal pain, or flatulenceafter the ingestion of milk or dairy products. In severe cases,malabsorption resulting from lactose intolerance may result in anorexiaand weight loss.

Lactose intolerance in humans can be treated in several ways.Prehydrolyzed milk and dairy products, having significantly reducedlevels of lactose, are commercially available. Supplements in the formof fungal or yeast-derived lactases that are added to lactose-containingliquids are also available. The lactase may also be in the form of atablet, which is ingested immediately before the consumption of milkproducts. Orally administrable tablets containing lactase derived fromAspergillus oryzae are available from Lactaid, Inc., Pleasantville, N.J.

A variety of factors influence the effectiveness of lactase-containingtablets in lactose intolerant patients. Tablets containing lactasederived from Aspergillus oryzae are intended to hydrolyze lactose in theacidic environment of the stomach. The empty stomach of a healthy humanhas a pH of 3 or less, but upon the ingestion of food, rises to about 5or 6. Merki, H. S. et al., “Pattern of 24 Hour Intragastric Acidity inActive Duodenal Ulcer Disease and in Healthy Controls”, Gut, 29, pp.1583-87 (1988). Lactases derived from Aspergillus oryzae have an optimumpH of approximately 5, and are therefore suitable for the hydrolysis oflactose in the stomach environment. Gekas, V. et al., “Hydrolysis ofLactose: A Literature Review”, Process Biochem, 20(1), pp. 2-12 (1985).

If a tablet containing lactase derived from Aspergillus oryzae isingested too far in advance of the consumption of lactose-containingfood, the lactase may be passed into the more neutral environment of theintestines, having a pH between about 6 and 8. Since the acid-activelactase is relatively inactive in this environment, the lactose may notbe enzymatically hydrolyzed, and the symptoms of lactose intolerance mayappear. Even if the lactase-containing tablet is ingested at the optimumtime before a meal, if a sufficiently large amount of lactose-containingfood products is consumed, some undigested lactose may pass to theintestines, resulting in discomfort in the lactose-intolerant patient.

Advances in medicine have increased the life expectancy of the Westernworld population. The elderly tend to have more neutral stomach pH dueto the increase incidence of achlorhydria. There is also an increase inthe use of H₂ blockers, which results in a more neutral stomach pH. As aresult of these trends, the pH profile of the stomach environment for asignificant portion of the population is moving out of the optimal rangefor enzymes derived from Aspergillus oryzae.

A need, therefore, exists for a composition which in addition tohydrolyzing lactose under the normal acidic conditions found in ahealthy stomach, has enzymatic activity in the more neutral environmentof the intestines and in the stomachs of the elderly and users of H₂blockers.

SUMMARY OF THE INVENTION

The present invention provides a composition for the enzymatichydrolysis of lactose. This composition contains a first, active lactasehaving a first optimum pH range and a second, active lactase having asecond optimum pH range. The first and second optimum pH ranges are of adifferent magnitude, which enables the composition to enzymaticallyhydrolyze lactose in environments having different or varying pHs. In apreferred embodiment, the first optimum pH range is about pH 3.0 toabout pH 6.0, while the second optimum pH range is about pH 6.0 to aboutpH 8.0. A process for enzymatically hydrolyzing lactose using the firstand second active lactases is also included in the present invention.

In a further embodiment of the present invention, a composition fortreating or controlling the symptoms of lactose intolerance is provided.This composition contains a first lactase having an optimum pH rangecorresponding to the pH of the stomach environment and a second lactasehaving an optimum pH range corresponding to the pH of the intestinalenvironment. The composition may further contain a pharmaceuticallyacceptable, orally administrable carrier material. A method for treatingor controlling the symptoms of lactose intolerance using theaforementioned composition is also disclosed.

The compositions and methods of the present invention provide for theenzymatic hydrolysis of lactose under the acidic conditions found in ahealthy, human stomach as well as in the more neutral conditions of theintestines and stomachs of H₂ blocker users and the elderly sufferingfrom achlorhydria.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is plot of hydrolytic activity versus pH for lactase derived fromAspergillus oryzae; and

FIG. 2 is a plot of hydrolytic activity versus pH for lactase derivedfrom K. lactis.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is characterized by the use of first and second,active lactases, having different optimum pH ranges, for the enzymatichydrolysis of lactose. This composition may be used to treat or controlthe symptoms of lactose intolerance in animals, particularly mammalssuch as humans.

As used in the present invention, “optimum pH range” means the pH overwhich the hydrolytic activity of the lactase is within about 10 to 100percent of its maximum, and “optimum pH value” means the pH at which thelactase exhibits maximum hydrolytic activity. FIGS. 1 and 2 show theoptimum pH ranges and values for lactase derived from A. oryzae and K.lactis, respectively. The reaction conditions for FIG. 1 were 2.0 μ/mL(micrograms/milliliter) A. oryzae lactase, 4.7% lactose, 30° C. and 10min. reaction. The reaction conditions for FIG. 2 were 200 μ/mL K.lactis lactase, 0.25% o-nitrophenyl-β-D-galactoside (substrate),phosphate buffer, 0.1 mM Mg⁺⁺, 37° C. and 15 min. reaction.

The first, active lactase is preferably an enzyme that is capable ofcatalyzing the hydrolysis of lactose in the stomach. Lactases derivedfrom fungi are generally known to have optimum pH values which fallwithin the acid range. Gekas, et al., supra, p. 3. The first, activelactase preferably has optimum pH range that encompasses pH 3.0 to aboutpH 6.0.

The first, active lactase can be derived from the following genera offungi: Aspergillus; Mucor; Fusarium; Scopuloriopsis; Alternaria; andCurvularia and the bacterium Thermus aquaticus. The lactases, having theoptimum pH value shown in the parentheses, are preferably derived fromthe following fungi: Aspergillus oryzae; (4.5-5.0) Aspergillus niger(3.0-4.0); Fusarium moniliforme (3.8-5.0); Scopulariopsis (3.6-5.0);Mucor pucillus (4.5-6), Alternaria alternara (4.5-5.5); and Curvulariainaegualis (3.4-4.3) and the bacterium Thermus aquaticus (4.5-5.5).

The second, active lactase has an optimum pH value which falls withinthe more neutral region. This lactase is capable of catalyzing lactosehydrolysis in the neutral environments of the intestines and thestomachs of the elderly, suffering from achlorhydria, and users of H₂blockers. The optimum pH range for these enzymes preferably encompassesabout pH 6.0 to about pH 8.0. Because of this activity in the neutralregion, the second, active lactase is capable of hydrolyzing anyundigested lactose which may be passed to the intestines, as well as anylactose in the stomach of a user having a more neutral stomach pH.

Lactases derived from yeast and bacteria are generally known to haveoptimum pH values in the more neutral region (6-7 and 6.5-7.5,respectively). Gekas, et al., supra, p. 2. The second, active lactasecan be derived from organisms within the genera of Kluyveromyces(Saccharomyces), Lactobacillus, Bacillus, Streptococcus, andEscherichia. Lactase derived from the following organisms, having theoptimum pH value shown in the parentheses, are preferred: Kluyveromyceslactis (6.5), Kluyveromyces fragilis (6.6), Lactobacillus thermophilus(6.2-7.1), Bacillus circulans (6.0), Lactobacillus bulgaricus (7.0),Leuconostoc citrovorum (6.5), Bacillus stearothermophilus (6.0-6.4),Streptococcus thermophilus (6.5-7.5), and Bacillus sp. (6.8).

In view of the above-noted article by Genkas, et al., which is herebyincorporated by reference, it will be appreciated that the lactases usedin the present invention can be produced by a variety of well knowntechniques. Many of these lactases are produced by commercial processeswhich cultivate the bacterium, yeast or fungus, and then isolate thelactase from the culture or culture broth of the microorganism. Furthertechniques for preparing such lactases may be found in U.S. Pat. No.3,629,073, issued Dec. 21, 1971; U.S. Pat. No. 3,718,739, issued Feb.27, 1973; and U.S. Pat. No. 3,919,049, issued Nov. 11, 1975, all ofwhich are hereby incorporated by reference.

In accordance with another embodiment of the present invention, thesecond lactase having an optimum pH range encompassing the neutralregion may contain an enteric coating. This coating remains intact inthe stomach, but will dissolve and release the second active lactaseonce it reaches the more neutral environment of the small intestine. Theenteric coating protects against irreversal deactivation of the lactasein the stomach, thus making the enzyme available for hydrolysis oflactose in the intestines. However, if the patient suffers fromachlorhydria, the second active lactase is generally not entericallycoated.

Suitable enteric coatings for the second active lactase include amyloseacetate phthalates, styrene-maleic acid copolymer, cellulose acetatesuccinate, cellulose acetate phthalate, polyvinyl acetate phthalate,hydroxypropylmethylcellulose phthalate, fatty acids, fatty acid esters,glycerol esters, polyglycerol esters, paraffin waxes, carnauba wax,formalized gelatin, shellac and hydrogenated vegetable waxes, such ashydrogenated castor oil and cottonseed oil. Other suitable entericcoatings are disclosed in Lieberman, H. A. et al., Pharmaceutical DosageForms: Tablets, Vol. 3, pp. 114-116 (1990), which is hereby incorporatedby reference.

The enteric coating is applied to the second lactase using conventionalparticle coating techniques. If the second lactase is granulated withother excipients, the resulting granule may also be coated with theenteric material. The enterically coated second lactase will generallycontain from about 2 to about 15 weight percent of the enteric coating.

If the composition is intended for use in patients having a more neutralstomach pH, a portion or all of the second lactase particles may be freeof the enteric coating. If only a portion of this enzyme in thecomposition is enterically coated, the uncoated enzyme is, uponingestion, immediately available for hydrolysis of lactose in thestomach, while the enterically coated enzyme is available for lactosehydrolysis in the intestines.

The lactase compositions of the present invention can be combined with apharmaceutically acceptable carrier and administered orally. The unitdosages of these compositions may be in the form of solid preparations,such as tablets, pills, capsules, caplets, powders, granules and wafers,or liquid preparations, such as suspensions or dispersions in aqueous ornon-aqueous vehicles, such as syrups and elixirs.

In preparing solid unit dosage forms, the first and second lactases aremixed with conventional solid fillers or carriers, such as starch, talc,calcium phosphate, calcium sulfate, calcium stearate, magnesiumstearate, stearic acid, sorbitol, mannitol, gelatin, natural orsynthetic gums, such as carboxymethylcellulose, methylcellulose,alginates, dextrans, acacia gum, karaya gum, locust bean gum, tragacanthand other conventional carriers. Additionally, other excipients such asdiluents, binders, lubricants, disintegrants, colors and flavoringagents may be employed.

Suitable liquid forms of the present invention can be prepared byincorporating the lactase in aqueous or non-aqueous dispersions,suspensions, or solutions. Conventional liquid carriers such asglycerol, and edible glycols, edible oils, such as cottonseed oil,soybean oil, corn oil, peanut oil, safflower oil, and other triglycerideoils, and dispersing or suspending agents, such as the aforementionednatural and synthetic gums.

Conventional methods are employed for preparing the solid and liquidforms of the present invention. Suitable techniques are described inRemington's Pharmaceutical Sciences, 18th Ed., Chapters 83 and 89(1990), which is hereby incorporated by reference.

The lactase compositions of the present invention can also be producedin powdered or granular form for direct admixture with food productsconsumed by subjects suffering from lactose intolerance. For instance,in the case of a lactose intolerant infant, a suitable amount of thelactase composition of the present invention, in a powdered or granularform, can be added directly to the milk or other food consumed by theinfant. In the case of an animal, such as a mammal, that normallyrequires a dietary regime of whey, the lactase composition of thepresent invention may be added directly to the whey.

The lactases employed in the compositions are present in therapeuticallyeffective amounts to hydrolyze the lactose normally present in the foodproducts consumed by the subject. This amount, of course, will varywithin wide limits, depending in part upon the lactase activity of theparticular enzyme, the magnitude of the lactose intolerance in theparticular subject and the dietary characteristics of the subject. Ingeneral, on an oral unit dosage basis for humans, the compositioncontains the first lactase in an amount equivalent to about 3000 toabout 6000 FCC Lac U and the second lactase in an amount equivalent toabout 7000 to about 35,000 neutral lactase units. A FCC lactase unit(FCC Lac U) and a neutral lactase unit are defined as that quantity ofenzyme that will liberate 1 μmol of o-nitrophenol fromo-nitrophenyl-β-D-galactoside per minute under the conditions, of theassay described in Food Chemicals Codex, National Academy Press, Wash.,D.C., pp. 491-2 (1981), which is hereby incorporated by reference, at pH4.5 and 6.5, respectively.

When the composition is in the form of a tablet or other solid form, aunit dosage will generally contain from about 1 to about 15 weightpercent of the first lactase, from about 8 to about 80 weight percent ofthe second lactase and from about 20 to about 80 weight percent of apharmaceutically acceptable carrier.

The lactase composition is administered to the subject prior to orconcurrently with the consumption of lactose-containing food products.

The following example illustrates a specific embodiment of the presentinvention. This invention, however, is not confined to the specificlimitations set forth in this example but rather to the scope of theappended claims. Unless otherwise stated, the percentages and ratiosgiven below are by weight.

EXAMPLE

This example provides a formulation for preparing a caplet form of thepresent invention containing an enzyme derived from Aspergillus oryzaeas the first lactase enzyme having activity in the acid region and anenzyme derived from Kluyveromyces lactis as the second lactase havingactivity in the neutral region. Each caplet has a total weight of 770 mgand an adult human would generally consume two or more caplets per dose.

The lactase powder derived from K. lactis is coated with the followingenteric suspension:

Ingredients % by Wt. Cellulose Acetate Phthalate NF 11.0 (AQUATERICPowder) Triacetin, USP 3.9 Polysorbate 80, NF 0.1 (TWEEN 80) PurifiedWater 85.0 100.0

The enzyme powder is charged into a Wurster fluidized bed coatingapparatus and fluidized by a flow of warm air. The enzyme powder attainsa product temperature of 28-37° C. The enteric suspension is thensprayed onto the fluidized enzyme particles at a rate of 9 mL/min. untilthe coated enzyme particles contain approximately 13% by weight of theenteric coating.

The enterically coated enzyme particles are combined with the followingingredients to produce the caplet:

Ingredients mg/Caplet % W/W Enterically Coated Lactase 517.6 75.0 (K.Lactis) Lactase Powder (A. oryzae) 15.0 2.2 Microcrystalline Cellulose,NF 153.4 22.2 Magnesium Stearate, NF 4.0 0.6 690.0 100.0

The enterically coated lactase (K. lactis), lactase derived from A.oryzae and microcrystalline cellulose are dry blended in a twin shellblender for 20 min. The magnesium stearate is added to the mixture andblended for an additional 5 min. The mixture is then compressed into acaplet on a rotary tablet press.

Various modifications can be made from the above-described embodimentswithout departing from the spirit and scope of the present invention.

What is claimed is:
 1. A solid, orally administrable composition for theenzymatic hydrolysis of lactose, comprising: from about 1 to about 15weight percent of a first, active lactase having a first optimum pHrange; from about 8 to about 80 weight percent of a second, activelactase having a second optimum pH range, wherein said first and secondoptimum pH ranges being different; and a solid pharmaceuticallyacceptable carrier.
 2. The composition of claim 1, wherein said firstoptimum pH range is within a pH range of about 3.0 to about 6.0.
 3. Thecomposition of claim 2, wherein said first, active lactase is a fungallactase or bacterial lactase derived from Thermus aquaticus.
 4. Thecomposition of claim 1, wherein said second optimum pH range is within apH range of about 6.0 to about 8.0.
 5. The composition of claim 4,wherein said second active lactase is a yeast or bacterial lactase. 6.The composition of claim 1, wherein said second active lactase isenterically coated.
 7. A solid, orally administrable composition fortreating or controlling the symptoms of lactose intolerance to humanscomprising: from about 1 to about 15 weight percent of a first, activelactase having an optimum pH range which is within the pH range of thestomach environment; from about 8 to about 80 weight percent of asecond, active lactase having an optimum pH range which is within the pHrange of the intestinal environment; and a solid, pharmaceuticallyacceptable, orally administrable carrier material.
 8. The composition ofclaim 7, wherein the optimum pH range of said first lactase is withinthe pH range of about 3.0 to about 6.0.
 9. The composition of claim 8,wherein said first lactase is a fungal lactase or a bacterial lactasederived from Thermus aquaticus.
 10. The composition of claim 9, whereinsaid first lactase is derived from the genera of fungi selected from thegroup consisting of Aspergillus, Mucor, Fusarium, Scopulariopsis, andAlternaria, and Curvularia.
 11. The composition of claim 10, whereinsaid first lactase is derived from a fungi selected from the groupconsisting of Aspergillus oryzae, Aspergillus niger, Fusariummonilforme, Scopulariopsis, Mucor pucillus, Alternaria alternara andCurvularia inaegualic.
 12. The composition of claim 11, wherein saidlactase is derived from a fungi selected from the group consisting ofAspergillus oryzae, Aspergillus niger and Mucor pucillus.
 13. Thecomposition of claim 7, wherein the optimum pH range of said secondlactase is within the pH range of about 6.0 to about 8.0.
 14. Thecomposition of claim 13, wherein said second lactase is a yeast orbacterial lactase.
 15. The composition of claim 14, wherein said secondlactase is derived from the genera selected from the group consisting ofKluyveromyces, Lactobacillus, Bacillus, and Streptococcus.
 16. Thecomposition of claim 15, wherein said second lactase is derived fromKluyveromyces lactis, Kluyveromyces fragilis, Lactobacillusthermophilus, Bacillus circulans, Lactobacillus bulgaricus, Bacillussp., Leuconostoc citrovorum, Bacillus stearothermophilus, andStreptococcus thermophilus.
 17. The composition of claim 16, whereinsaid second lactase is Kluyveromyces lactis.
 18. the composition ofclaim 17, wherein said second lactase is enterically coated.
 19. Thecomposition of claim 7, in a unit dosage form comprising an amount ofsaid first lactase equivalent to about 3000 to about 6000 FCC Lac U andan amount of said second lactase equivalent to about 7000 to about35,000 neutral lactase units.
 20. A solid, oral dosage form for treatingor controlling the symptoms of lactose intolerance in humans,comprising: an amount of a first, active lactase having a first optimumpH range equivalent to about 3000 to about 6000 FCC Lac U; an amount ofa second, active lactase having an optimum pH range equivalent to about7000 to about 35,000 neutral lactase units; and a solid,pharmaceutically acceptable carrier.